Combining Residue Substitution and Deletion Engineering Enhances the Substrate Acceptance of Ene-Reductase for Highly Enantioselective Synthesis of β-Cyano Ester Scaffolds

Chiral β-cyano ester scaffolds are highly valuable synthons in pharmaceutical synthesis. The direct asymmetric hydrogenation of β-cyanoacrylates, catalyzed by ene-reductases (ERs), offers an efficient route to these scaffolds with a high atom economy under mild reaction conditions. However, the narrow substrate acceptance of ER reported in the previous work has limited its application in asymmetric hydrogenation. In this study, we engineered the substrate-binding pocket of an ER from Saccharomyces eubayanus (SeER) to access a panel of chiral β-cyano ester scaffolds with broad structural diversity. Using residue-deletion engineering, mutant M1 was generated and exhibited 416-fold higher catalytic activity toward the model substrate compared to the wt-SeER, along with improved enantioselectivity (from rac to 97% ee). The optimal mutant M4, obtained through saturation mutagenesis and iterative combination, exhibited 1.36 × 104-fold higher catalytic efficiency than that of wt-SeER, and the enantioselectivity further increased to 99.7% ee. Mutant M4 demonstrates broad substrate acceptance and was successfully applied in a gram-scale (200 mM) asymmetric synthesis of Pregabalin (a first-line antiepileptic agent) with 99.7% ee and 65% isolated yield. This study highlights residue deletion as an effective strategy for modifying the ER binding pocket to access the asymmetric hydrogenation of β-cyanoacrylates but also provides valuable engineering guidance for enhancing the substrate acceptance of other ERs possessing similar pockets.